A pilot study on the efficacy of a seaweed mud application in the treatment of cellulite

Cellulite represents a common multi‐factorial condition that affects nearly all women and is now recognized as a clinical condition associated with systemic factors and negative psychological effects. Several noninvasive and minimally invasive treatments were developed during the last few years, but limited evidence supports many of them due to lack of evidence, insufficient participants, and potential adverse effects.


| INTRODUC TI ON
Cellulite is a widely spread condition that affects between 80% and 90% of post-pubertal women and is also present in males. 1 Although previously regarded as a harmless skin condition primarily affecting aesthetics, cellulite is now recognized as a clinical condition associated with systemic factors and has been shown to cause negative psychological effects in patients. 2The thighs, hips, and buttocks are the most common areas affected by cellulite, which is characterized by the presence of skin dimpling.The complete understanding of cellulite's physiology is yet to be fully clarified.However, several experimental evidence suggest that cellulite is a complex process involving multiple factors that may have an impact, to varying degrees, on cellular and non-cellular components of the skin and subcutaneous tissues in an asynchronous, chronic, and unpredictable manner, like a chronic progressive degenerative condition. 3,4Indeed, the etiology and exacerbation of cellulite have been associated with chronic inflammation, suggesting its significance as a risk factor.
Notably, it has been shown that cellulite can be accompanied by chronic inflammation that alters local tissue, causing skin atrophy and adipolysis. 5,6In the pathogenesis of cellulite, mesenchymal stem cells (MSCs), characterized by their abundance of estrogen receptors, may play a pivotal role.These cells are mobilized in response to various stressors, including oxidative stress, extracellular matrix remodeling, and the generation of reactive oxygen species (ROS).
The interplay between chronic inflammation and oxidative stress is further substantiated by the observation that the absence of antioxidant defense mechanisms is associated with an enhanced inflammatory phenotype.Conversely, the upregulation of ROS-producing enzymes also contributes to the inflammatory state. 7ere have been studies on various noninvasive techniques, including massage, cosmeceuticals, and laser therapy, as well as minimally invasive techniques such as subcision and collagenase injection, to improve cellulite. 2,8However, limited evidence supports many of these treatments due to the lack of a validated and convenient tool for evaluating cellulite severity or an insufficient number of study participants.Additionally, some of these procedures may lead to adverse effects including scars and keloid formation.
Among non-invasive techniques, cosmetic and/or cosmeceutical treatments are commonly employed for cellulite reduction.
Cosmeceuticals are topical products that occupy a niche between cosmetics and pharmaceuticals, aiming to improve both the health and aesthetics of the skin. 9,10Their primary challenge is to transport the active ingredients to their target in sufficient concentration to have a therapeutic effect. 11Over-the-counter gels or creams, frequently utilized to mitigate cellulite, typically comprise caffeine, methylxanthines, retinol, ascorbic acid, alpha-bisabolol, papaya extract, other plant extracts, vegetable oils, urea, and squalene.Seaweed, in particular, is recognized as a valuable source of functional foods and innovative cosmeceutical compounds.Consequently, research over the past two decades has extensively explored the bioactive effects of seaweeds on skin health.Seaweeds, especially brown seaweeds, produce both primary and secondary metabolites, including polysaccharides, proteins, polyphenols, carotenoids, and phytosterols, that demonstrate different beneficial activities on biological systems.[14][15][16][17][18] However, very few studies have explored the effect of seaweeds on cellulite. 19Accordingly, the present study aimed to assess the effectiveness and tolerability of a cosmetic mud containing seaweed in improving tissue alterations characteristic of cellulite, using both clinical and instrumental measurements over a 4-week treatment period.

| Patients' population
A group of 60 female patients, ranging in age from 32 to 71 years, participated in this clinical study.The participants were selected from 92 female patients.The inclusion criteria included being at least 18 years of age, female, and providing consent to participate in the study.All enrolled patients had clinically detected cellulite on their gluteal and posterior thighs.Patients who had received any aesthetic treatments within the 6 months before the study, those with a history or current diagnosis of heart, thyroid, or metabolic disorders, tumors, or immunosuppression, those with a body mass index (BMI) greater than 30, and pregnant or lactating patients were excluded from the study.All patients successfully attended all scheduled visits.

| Patient written informed consent and ethical approval
Each patient provided written informed consent before the study, which adhered to the principles outlined in the Declaration of Helsinki.

| Study protocol
Participants underwent application for 4 weeks (three applications of 45 minutes in the first week, two in the second and third weeks, and one in the fourth week) of a commercially available cosmetic seaweed mud (Fanghi d'alga GUAM® F.I.R., Lacote s.r.l., San Prospero, Modena, Italy) on the buttocks and posterior thighs.The seaweed mud is composed of over 15% dried brown seaweed (Laminaria digitata), 6% phyto-extracts of brown seaweed (Laminaria digitata and Fucus vesiculosus), clay, and black tourmaline for the remaining percentage.A minimal percentage of other extracts are included for their fragrance.
One kilogram of seaweed mud was used per application.The product was stirred prior to use and applied with a gentle massage onto the designated treatment areas that were wrapped with transparent film, as reported in Figure 1.After 45 min, the seaweed mud was rinsed with fresh water.Before the initial application (baseline) and after the 4week treatment period, morphometrics, dermatology, instrumentation analyses, and histological evaluations were conducted.Patient satisfaction was assessed using a 5-point Likert-scale questionnaire.

| Physical examination
The initial physical examination was carried out on each patient before the treatment with seaweed mud, as previously described. 20e examination included measurements of weight, height, BMI, and various circumferences such as waist, belly, hips, upper thigh, and median thigh.

| Clinical and dermatological evaluation
The patients underwent a total body skin examination in a standing position with relaxed muscles.The examination assessed the following parameters: the presence of paresthesia, telangiectasias, varices, skin stretch marks, scars, positive ulnar fovea sign test, pain at the tissue pinch test, malleolar edema, and heaviness in the lower limbs.
The color of the trochanteric region, which is commonly affected by cellulite, was also evaluated.The color could be unchanged, exhibit a yellowish-gray tone, or display skin pallor.Following the dermatological examination, the patient's skin hydration and elasticity levels were measured (SoftPlus, Callegari, Parma, Italy).Additionally, ultrasonography of the trochanteric region of the thighs was performed using the MyLab™ Gamma linear probe (Esaote Touch, Genova, Italy) with a frequency of 12 MHz.Each patient was evaluated at both the beginning and conclusion of the last treatment (4 weeks).

| Histological analysis
Local anesthesia was administered prior to performing a biopsy on the skin affected by cellulite.The biopsies were obtained from the trochanteric region using a specific biopsy punch (Kai Medical, Oyama, Japan), with a diameter of 4.0 mm and a length of 13.0 mm.Sample preparation and histological analysis with hematoxylin and eosin (H&E) staining were performed as previously described. 21rthermore, two distinct H&E-stained fields at 40× magnification (1 mm 2 ) were selected for the inflammatory cell count for each patient.

| Statistical analysis
GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA, USA) was utilized for statistical analyses.The normality of the data distribution was evaluated using the Shapiro-Wilk normality test.For normally distributed data, comparisons were made using the paired parametric Student's t-test.In cases where data did not follow a normal distribution, the Wilcoxon matched-pairs signed rank test (two-tailed) was used.Results are presented as the arithmetic mean ± standard deviation (SD).A significance level of p < 0.05 was considered to indicate statistical significance.

| Physical and anthropometric examination
A total of 60 female patients (mean age of 50.20 ± 11.15 years) were included in the study (Table 1).In detail, the age distribution of the patients was as follows: 15 patients between 30 and 39 years, 18 patients between 40 and 49 years, 16 patients between 50 and 59 years, and 11 patients over 60 years.Among these, 22 women were in menopause (36.7%) and 39 had a history of at least a pregnancy to term (65.0%).The average height was 162.9 ± 5.4 cm, the mean weight was 67.7 ± 6.8 kg, and the average BMI was 25,1 ± 2.1 kg/m 2 .As part of the physical examination, the circumferences of the waist, belly, hips, and upper and median thighs of all patients were evaluated at baseline and after 4 weeks of treatment.
A significant reduction of all circumferences analyzed was observed (Figure 2).No adverse events were reported during or following the treatment period, including interference with normal thyroid  2.

| Clinical and dermatological evaluation
In the skin evaluation, it was discovered that 54 patients (90%) exhibited noticeable orange peel skin, while six patients (10%) showed visible alterations following the pinch test.Among the participants, 15 patients (25%) experienced pain during the pinch sensation.Upon examining the affected anatomical sites for cellulite, three patients presented with pale skin, and four patients exhibited a yellowish-gray tone.Additionally, one of these patients also suffered from severe venous insufficiency.Furthermore, skin hydration and elasticity levels were significantly higher after the 4-week treatment with seaweed mud, indicating an overall improvement in tissue properties (Table 3).
Ultrasound observations confirmed that cellulite lesions exhibited a rich echo pattern in the epidermis and dermis, while the adipose protrusions in the subcutis displayed a low echo pattern and were scattered with hyperechoic connective shoots. 21Before the treatment, the common pattern was characterized by an increased thickness of the skin and subcutaneous layers with a low-echo signal, the presence of inflammation, and a reduction in vascularization.
Additionally, low-echo nodules and fascial bands were observed in most patients.The predominant feature in all patients was the obliquely oriented fascial band, which was observed in great detail.
Concurrently, a difference in band orientation was detected in most patients (Figure 3A).Results demonstrated that, compared with baseline, the 4-week treatment with seaweed mud resulted in a significant decrease in adipose tissue thickness, improvement of vascularization, and re-organization of the cutaneous and sub-cutaneous architecture due to the induction of collagen synthesis (Figure 3B).

| Histological analysis
Before treatment, histological analysis of stained slides at 10×, 20×, and 40× magnification revealed the presence of various histological features associated with cellulite in most patients.In the epidermis, peculiar changes in cell replication at the baseline and islands of keratinocyte hypertrophy at the stratum corneum were observed.The dermis exhibited alterations in the quality of hyaluronic acid and a disruption in the structural architecture of collagen and elastic fibers.
Other characteristics of the dermis included chronic inflammation with reactive fibrosis, changes in microcirculation, the presence of edema in the reticular dermis, thickening of connective fibers, and adipocytes that appeared distinct in shape from subcutaneous adipocytes.The hypodermis displayed anisopoikilocytosis, fibrotic thickening of collagen surrounding the lobules, a concentric arrangement of adipose cells contributing to the formation of micronodules, and alterations in the microcirculation of arterioles and venules (Figure 5A-G).
After the 4-week treatment with seaweed mud, many of the peculiar histological features of cellulite disappeared, with an overall restoration of normal tissue architecture (Figure 5H-K).Histological analysis of cellulite biopsies showed a significant improvement in dermal organization with the induction of collagen synthesis, the structural architecture of the extracellular matrix, and microcirculation.In parallel, reduction in chronic inflammatory infiltrate, lipid deposition, reactive fibrosis, and edema were also observed.
In detail, pre-treatment tissues were characterized by a rich concentration of chronic inflammatory lymphocytes (35.89 ± 7.18 cells per mm 2 ) mainly localized around the vascular structures and the collagen fibers.These cells were significantly reduced and sometimes absent (6.55 ± 3.05 cells per mm 2 , p < 0.0001) in the post-treatment histological samples, suggesting an inflammation-inhibiting effect.
Rare histiocytes (<5 cells per mm 2 ) were identified with no significant differences between pre-and post-treatment samples.No accumulation of neutrophilic or eosinophilic granulocytes in pre-and post-treatment samples was detected, excluding tissue necrosis and allergic phenomena, respectively (Figure 5).

| D ISCUSS I ON AND CON CLUS I ON S
Seaweeds are recognized sources of important bioactive components such as polysaccharides (e.g., alginate, fucoidan), proteins (e.g., phycobiliproteins), polyphenols (e.g., phlorotannins), carotenoids TA B L E 2 Detailed statistics (GraphPad Prism software v.8) about differences of circumferences of waist, belly, hips, and upper and median thigh at baseline and after 4 weeks of treatment with seaweed mud.
Frontal and (B) posterior view of a patient during the treatment with seaweed mud.
activity.No reddening, swelling, or irritation was observed around treatment areas at the time of testing.Overall, patients reported experiencing only a slight, non-painful tingling sensation during the treatment.A 5-point Likert scale questionnaire assessed subjective satisfaction.With a score of 3.68 ± 0.6, patients expressed satisfaction with the results of the treatment.Details and statistics of all anthropometric evaluations are reported in Table

1 F I G U R E 2
Moreover, a clear correlation was found between the severity of cellulite and the increased presence of adipose protrusions in the TA B L E 1 Characteristics of female patients analyzed in this study.Differences of circumferences of waist, belly, hips, and upper and median thigh at baseline and after 4 weeks of treatment with seaweed mud; **** = p < 0,0001 (GraphPad Prism software v.8).ultrasound images.The significant reduction of low-echo subcutaneous micronodules after a 4-week treatment with seaweed mud suggests a reduction in the severity of cellulite (Figure4).

Circumferences Baseline (cm) After 4 weeks treatments (cm) Difference between means (cm) 95% confidence interval p Value
Note: Data are represented as the arithmetic mean ± SD.